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MOLECULAR PHARMACOLOGY, 56:886 –894 (1999).
Calcineurin Enhances Acetylcholinesterase mRNA Stability
during C2-C12 Muscle Cell Differentiation
Z. DAVID LUO, YIBIN WANG, GUY WERLEN,1 SHELLEY CAMP, KENNETH R. CHIEN, and PALMER TAYLOR
Departments of Pharmacology (Z.D.L., G.W., S.C., P.T.) and Medicine (Y.W., K.R.C.), University of California-San Diego, La Jolla, California
Received April 1, 1999; accepted August 5, 1999
Differentiation of skeletal muscle from myoblasts to myotubes is globally controlled by the expression of muscle-specific genes of the myo D family. However, following initiation
of differentiation, expression of genes encoding proteins important for controlling cellular excitability, such as acetylcholinesterase (AChE) and nicotinic acetylcholine receptors
(nAChR), occurs through distinct mechanisms. Although the
increased expression of nAChR arises from enhancement of
its transcription rate (Evans et al., 1987; Baldwin and Burden, 1988), the expression of AChE appears mainly due to
stabilization of a labile AChE mRNA (Fuentes and Taylor,
1993; Luo et al., 1994).
Previous studies indicated that regulation of intracellular
Ca21 through L-type Ca21 channels in the plasma membrane or intracellular ryanodine-sensitive Ca21 channels
This study was supported by National Institutes of Health (Grants GM
18360 to P.T., F32HL09848 to Z.D.L., and HL46345 to K.R.C.) and a grant
from the Muscular Dystrophy Association to P.T. G.W. was supported by the
Swiss Academy of Medical Sciences and the Swiss National Science Foundation (Grant 823A-046706). The Basel Institute for Immunology was founded
and is supported by F. Hoffman-La Roche Ltd., Basel, Switzerland.
1
Current address: Basel Institute for Immunology, Grenzacherstrasse 487,
CH-4005, Basel, Switzerland.
FKBP12, immunophilins known to be intracellular-binding targets for CsA and tacrolimus, respectively. However, cellular
levels of calcineurin, a calcium/calmodulin-dependent phosphatase known to be the cellular target of ligand-immunophilin
complexes, increase 3-fold during myogenesis. Overexpression of constitutively active calcineurin in differentiating cells
reduces AChE mRNA levels and CsA antagonizes such an
inhibition. Conversely, overexpression of a dominant negative
calcineurin construct increases AChE mRNA levels, which are
further enhanced by CsA. Thus, a CsA sensitive, calcineurin
mediated pathway appears linked to differentiation-induced
stabilization of AChE mRNA during myogenesis.
plays an important role in stabilization of AChE transcripts
during muscle development in mouse C2–C12 myocytes (Luo
et al., 1994) and in intact mouse skeletal muscle (Luo et al.,
1996). Other studies revealed that immunosuppressant cyclosporin A (CsA) regulates mRNA stability of interleukin-3
(Nair et al., 1994). Furthermore, FKBP12, an intracellular receptor for another immunosuppressant tacrolimus (FK506),
was copurified with ryanodine receptors (Jayaraman et al.,
1992) and found to modulate intracellular calcium release by
stabilizing ryanodine-sensitive calcium channels in skeletal
muscle (Timerman et al., 1993; Brillantes et al., 1994).
CsA and tacrolimus bind to intracellular immunophilins
such as cyclophilin A and FKBP12, respectively (Handschumacher et al., 1984; Harding et al., 1989; Standaert et al.,
1990). The immediate cellular target for the complexes of
CsA-cyclophilin and FK506-FKBP12, but not for the complexes of CsA-FKBP12 or FK506-cyclophilin, is calcineurin, a
Ca21/calmodulin-dependent protein phosphatase (Liu et al.,
1991). The influence of CsA and tacrolimus on regulation of
mRNA stability in other systems and the linkage of their
intracellular receptors to intracellular Ca21 mobilization
prompted us to investigate the functional role of the cal-
ABBREVIATIONS: AChE, acetylcholinesterase; nAChR, nicotinic acetylcholine receptor; CsA, cyclosporin A; CsH, cyclosporine H; BCA, bicinchoninic acid; a-BTX, a-bungarotoxin; HA, hemagglutinin; DFP, diisopropyl fluorophosphate; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; b-gal, b-galactosidase; CnADCaM-AI, constitutively active calcineurin catalytic construct; DRB, 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole; BKO, B subunit knock out construct; Adv, adenovirus.
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ABSTRACT
Treatment of C2–C12 mouse myoblasts with the immunosuppressant drug cyclosporin A (CsA) enhances the increase in
acetylcholinesterase (AChE) expression observed during skeletal muscle differentiation. The enhanced AChE expression is
due primarily to increased mRNA stability because CsA treatment increases the half-life of AChE mRNA, but not the apparent transcriptional rate of the gene. Neither tacrolimus (FK506),
an immunosuppressive agent with a distinct structure, nor cyclosporine H, an inactive congener of CsA, alters AChE expression. The enhanced AChE expression is associated with the
muscle differentiation process, but cannot be triggered by CsA
exposure before differentiation. Myoblasts and myotubes of
C2–C12 cells express similar amounts of cyclophilin A and
This paper is available online at http://www.molpharm.org
Calcineurin and Acetylcholinesterase mRNA Stability
cineurin-mediated pathway in regulation of AChE mRNA
stabilization during myogenesis.
Experimental Procedures
Determination of AChE Activity. AChE was extracted from
rinsed C2–C12cells in 0.01 M sodium phosphate buffer containing 1
M NaCl, 1% Triton X-100, 0.01 M ethylene glycol bis(b-aininoethyl
ester)-N,N,N9,N9-tetraacetic acid, and a spectrum of protease inhibitors, and protein concentrations were determined with BCA reagents. AChE in the media was concentrated .10-fold in Centriprep
30 concentrators (Amicon Corp., Beverly, MA). Enzyme activity was
determined at room temperature as described by Ellman et al. (1961)
in 0.1 M sodium phosphate buffer (pH 7.0) containing 0.3 mM 5,59dithiobis-(2-nitrobenzoic)acid, 0.5 mM acetylthiocholine iodide, and
0.05 to 0.1 ml of cell extract or concentrated medium. More than 90%
of the enzyme activity is due to AChE as determined from the
presence of 10 mM BW284c51 (data not shown).
Determination of Creatine Kinase Activity. Rinsed C2–C12
myotubes differentiated for 3 days were extracted in PBS containing
0.5% Triton X-100 and a spectrum of protease inhibitors. After sonication and centrifugation, supernatants were assayed for protein
concentrations with BCA reagents and for creatine kinase activity
with Sigma Diagnostics Procedure 520.
b-Galactosidase (b-Gal) Staining. C2–C12 cells infected with
adenovirus containing the lac-Z gene were stained for b-gal activity
as described by Sanes et al. (1986). Briefly, infected cells were rinsed
with PBS and fixed for 5 min on ice in PBS containing 2% formaldehyde and 0.2% glutaraldehyde. After washing, the cells were overlaid with a PBS reaction mixture containing 1 mg/ml 4-Cl-5-Br-3indolyl-b-galactosidase, 5 mM potassium ferricyanide, 5 mM
potassium ferrocyanide, and 2 mM MgCl2 and incubated at 37°C
until desired color intensity was achieved. The cells were then fixed
for 10 min at room temperature, rinsed, and stored in PBS.
Determination of nAChR Expression. Densities of cell surface
nAChR were monitored by binding of [a-125I]BTX to intact cells
cultured in six-well plates at room temperature. Cells were washed
with differentiation medium and incubated for 10 min in the presence or absence of 10 mM carbamylcholine chloride. [a-125I]BTX at a
final concentration of 10 nM was added directly to each well and
incubated for 2 h. Cells were washed three times gently with K1Ringer’s buffer (140 mM KCl, 5.4 mM NaCl, 1.8 mM CaCl2, 1.7 mM
MgCl2, 25 mM HEPES, 0.03 mg/ml bovine serum albumin, pH 7.4)
and 1 ml of 1 M NaOH was added to each well to lyse the cells. Cell
lysates were counted in a gamma counter and protein was assayed in
the cell lysates with BCA reagents.
Run-On Transcriptional Analysis. Nuclei were isolated from
cultured cells and stored at 270°C as described previously (Luo et
al., 1994). Nuclei (200 ml) were thawed and mixed with equal volume
of 23 buffer containing 10 mM Tris-HCl, pH 8.0; 5 mM MgCl2; 0.3 M
KCl; 5 mM dithiothreitol; 1 mM each of ATP, GTP and CTP; and 10
ml of 32P-a-UTP. Radiolabelled mRNA was transcribed, isolated, and
hybridized for at least 36 h at 65°C to slot blots containing 5 mg of
each plasmid DNA. A 1.7-kilobase cDNA fragment within the coding
region of mouse g-nAChR subunit in a pSP-65 vector was linearized
with BamHI. A 1.4-kilobase cDNA fragment of mouse a-tubulin in
Bluescript SK II1 was linearized with KpnI. Bluescript SK II1 plasmid DNA linearized with EcoRI or KpnI was used as control for the
dsDNAs. For detecting AChE mRNA, control M13 phage DNA and
that containing a 2.3-kilobase single-strand antisense AChE cDNA
insert were used. After extensive washing with 23 standard saline
citrate buffer, radioactivity was determined by autoradiography and
densitometry.
Determination of AChE mRNA Stability. C2–C12 cells differentiated for 3 days in the presence or absence of 1 mM CsA were
treated with 30 mg/ml 5,6-dichloro-1-b-d-ribofuranosylbenzimidazole
(DRB) to block transcription and total RNA was extracted at the
designated time after treatment. The decay rate of AChE mRNA was
then determined by RNase protection with 50 mg of total RNA per
lane. The AChE mRNA half-lives calculated from these data could be
overestimated because slight reductions in the amount of total RNA
occurred at longer times after DRB treatment; use of a constant
amount of total RNA per sample contributes to the relatively high
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Materials. CsA and cyclosporine H (CsH) were from Sandoz Ltd.
(Basel, Switzerland). A stock solution of CsA and CsH was dispensed
in ethanol and Tween 80 followed by addition of PBS. Tacrolimus
was from Fujisawa Ireland, Ltd. (Kerry, Ireland). Ethanol stock
solutions were further diluted into culture medium at the time of
treatment. Bicinchoninic acid (BCA) protein assay reagents were
from Pierce Chemical Co. (Rockford, IL). The creatine kinase assay
materials and all other chemicals were from Sigma Chemical Co. (St.
Louis, MO). Components of culture medium were from Gibco Laboratories (Grand Island, NY). [a-125I]Bungarotoxin ([a-125I]BTX; specific activity 14.5 mCi/mg) was from NEN Research Products (Wilmington, DE). 32P-a-UTP (specific activity ;800 Ci/mmol) was from
Amersham Corp. (Arlington Heights, IL). The polyclonal anticyclophilin A antibody was from Affinity BioReagents, Inc. (Neshanic
Station, NJ), the monoclonal anticalcineurin antibody was from
Transduction Laboratories (Lexington, KY), the anti-FKBP12 antibody was a gift from Dr. Steven J. Burakoff at the Dana-Farber
Cancer Institute (Boston, MA) and the antihemagglutinin (HA) antibody was a gift from Dr. Michael Karin at the University of California-San Diego. The secondary antibody and detection reagents
were from Amersham Corp. (Buckinghamshire, England). Tris-glycine polyacrylamide gels were from NOVEX (San Diego, CA).
Tissue Culture. C2–C12 cells (American Type Culture Collection, Rockville, MD) were stored at 270°C and cultured at 37°C, with
5% CO2 in Dulbecco’s modified Eagle’s medium containing 20% fetal
bovine serum; 0.5% chick embryo extract; and 1% penicillin, streptomycin, and amphotericin B stock solution (Antibiotic-Antimycotic;
Gibco Laboratories). Cells were passed either two or three times
before plating. Differentiation from myoblasts to myotubes was induced at ;70% confluence by replacing the high serum medium with
Dulbecco’s modified Eagle’s medium containing 2% horse serum and
1% of the above-mentioned antimicrobial stock solution. To measure
secreted AChE in the medium, cells were differentiated in medium
containing serum pretreated with 0.1 mM diisopropyl fluorophosphate (DFP) to inhibit serum esterase activity. The DFP-treated
serum was incubated overnight at room temperature, sterile filtered
and held for at least 48 h at 4°C, and then assayed for residual DFP
and AChE activity. Such treatment results in .99% inhibition of
serum esterase activity with no inhibitory activity toward exogenously added AChE activity (Coleman and Taylor, 1996).
RNA Extraction and RNase Protection Assay. Total RNA was
extracted from cultured cells with TRIzol reagent from Gibco Laboratories and stored at 220°C. mRNAs encoding AChE and the g-subunit of nAChR (g-nAChR) were quantified by RNase protection as
described in Luo et al. (1998). The antisense probe of AChE mRNA
was made from a mouse Ache cDNA subcloned in Bluescript SK II
plasmids and linearized with XhoI. This probe allows us to distinguish exon 4 to 6 spliced AChE mRNA from other splice variants. A
fully protected probe in RNase protection assays indicates the presence of exon 4 to 6 spliced AChE mRNA, whereas the two shorter
protected fragments represent other splice variants (Luo et al.,
1998). A probe made from a 1.7-kilobase mouse nAChR g subunit
cDNA (kindly provided by Drs. Jim Boulter and Stephen Heinemann, Salk Institute, San Diego, CA), cloned in pSP-65 and linearized with XhoI, was used to hybridize with mRNA of the g-nAChR
subunit. To normalize for sample loading, a probe was made from a
316-bp cDNA fragment of the mouse glyceraldehyde 3-phosphate
dehydrogenase (GAPDH) from Ambion, Inc. (Austin, TX). Molecular
masses of the protected probes were estimated by electrophoresis on
polyacrylamide gels. Densities of bands were quantified by densitometry (UltroScan XL; Pharmacia LKB Biotechnology Inc., Picataway, NJ) and standardized by ratios to the GAPDH bands.
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Infection of Calcineurin-Containing Recombinant Adenoviruses in C2–C12 Myocytes. Different dosages of adenovirus were
added into culture media to achieve specified multiplicity of infection. The optimal time of infection during muscle differentiation was
determined by detecting HA peptide expression in C2–C12 cells
infected with pAdv/HA-CnADCaM-AI between day 0 and day 2.
Differentiating myocytes were infected more efficiently than undifferentiated myoblasts (see Results), consistent with findings reported in other rodent muscle cell lines (Quantin et al., 1992). In our
experiments, cells infected at day 1 of differentiation express the
largest amounts of HA peptides (see Results). This may result from
two factors. First, expression of integrin receptors, essential for the
attachment of adenovirus to host cells (Kohout et al., 1996), is developmentally regulated in C2–C12 cells (Collo et al., 1993; Ziober
and Kramer, 1996). The difference in infection efficiencies may result
from different expression levels of integrin receptor subtypes mediating the adenovirus infection. Second, when cells were infected at
later stages of differentiation (day 2 or 3) and harvested 2 days later,
some mature infected myotubes may have detached and died. Thus,
cells at day 1 of differentiation were used for all the infection experiments. Adenovirus vector expressing b-gal was used in parallel
infections (Wang et al., 1998). After 16 h of infection, medium was
removed and cells were incubated for an additional 2 days in fresh
medium before extraction of total RNA.
Statistical Analyses. Unpaired Student’s t tests were performed
where significance is indicated by a two-tailed P value , .05.
Results
Enhancement of AChE Expression by CsA in C2–C12
Cells during Myogenesis. CsA treatment caused concentration-dependent increases in the expression of AChE in
differentiating C2–C12 cells (Fig. 1). The half-maximal enhancement occurred at ;300 nM, a concentration that correlates well with the Kd value in binding studies (200 nM;
Fig. 1. Concentration dependence of immunosuppressants on differentiation-induced AChE expression in C2–C12 cells. C2–C12 myoblasts at
;70% of confluence were differentiated into myotubes for 3 days in low
serum medium in the presence or absence of CsA, CsH, or tacrolimus.
AChE was extracted and activity was determined by the method of
Ellman et al. (1961). Data are presented as percentage of control within
each experiment and the values reported are means 6 S.E. from at least
six independent determinations.
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values in the later time points of the curves (see Results). A GAPDH
antisense probe was included in all the experiments for normalization of sample loading for each time point.
Western Blots. To examine the cellular levels of immunophilins
or calcineurin, C2–C12 cells were washed three times with PBS and
extracted in 50 mM Tris-HCl buffer, pH 8.0, containing 150 mM
NaCl, 0.5% Triton, 1 mM EDTA, and mixture of protease inhibitors.
After removal of an aliquot for protein assay, the extracts were
subjected to polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes (Schleicher & Schuell, Inc.,
Keene, NH) electrophoretically. After blocking nonspecific binding
sites with 5% low-fat milk in PBS containing 0.1% of Tween-20,
antibodies against specified proteins or peptides were used to blot
the membrane in the same buffer for 1 h at room temperature. After
washing the nitrocellulose membrane two times with the same
buffer and one time with a buffer containing 150 mM NaCl and 50
mM Tris-HCl (pH 7.5), the antibody-protein complexes were blotted
for 1 h at room temperature with secondary antibodies labeled with
horseradish peroxidase in the buffer containing 5% low-fat milk.
After extensive washing, the protein-antibody complexes were detected with chemiluminescent reagents (Amersham Corp.).
Construction of Recombinant Adenoviruses Containing
Calcineurin cDNAs. Conventional transfection methods, such as
calcium phosphate or lipofectamine-mediated transfection, resulted
in low transfection efficiencies for C2–C12 cells. More importantly,
the transfected cells lost CsA responsiveness (Z.D.L., unpublished
data), presumably due to transient permeabilization and influx of
Ca21. To overcome the transfection limitations, adenoviruses containing either the constitutively active or the dominant negative
recombinant calcineurin constructs were used to infect these cells.
The constitutively active construct encodes a truncated calcineurin
catalytic subunit (CnADCaM-AI) that lacks the sequences encoding
the functional calmodulin-binding and autoinhibitory domains
(O’Keefe et al., 1992). This construct was designed to mimic proteolysed forms of calcineurin known to have constitutive calcium-independent phosphatase activity in vitro (Hubbard and Klee, 1989;
Werlen et al., 1998). Overexpression of this truncated calcineurin
subunit in human Jurkat cell line also exhibits Ca21-independent,
constitutive phosphatase activity (O’Keefe et al., 1992; Werlen et al.,
1998).
The dominant negative construct lacks the autoinhibitory domain,
calmodulin-binding domain, and most of the catalytic core, but contains the binding domain for the B (regulatory) subunit (Muramatsu
and Kincaid, 1996). Overexpression of this “B-subunit knock out”
(BKO) construct in Jurkat cells results in sequestration of the endogenous B subunit and suppression of calcineurin-mediated activity
(Muramatsu and Kincaid, 1996).
Plasmids of pAdv/HA-CnADCaM-AI and pAdv/HA-BKO were
constructed by inserting an EcoRI fragment containing HACnADCaM-AI or HA-BKO coding sequences from the pSRa3/HACnADCaM-AI or pSRa3/HA-BKO plasmids, respectively, into the
EcoRI site of the pAC/CMV vector. The HA epitope was used to
detect overexpression of these constructs in Western blots because
commercially available anticalcineurin A antibodies recognize an
epitope in the deleted region of these constructs and therefore were
not useful in our study. The HA tag at the N terminus does not affect
the function of the constructs (data not shown) (Werlen et al., 1998).
These recombinant adenoviruses were generated through homologous recombination between cotransfected pJM17 plasmid (Wang et
al., 1996) and pAdv/HA-CnADCaM-AI or pAdv/HA-BKO in 293 cells
(American Type Culture Collection). The resulting adenoviruses
were plaque purified and amplified in 293 cells. The genomic structure of the recombinant adenovirus was confirmed by polymerase
chain reaction analysis with oligonucleotides specific for the insertion site at the E1a region. Recombinant adenoviruses were prepared
from CsCl density gradient ultracentrifugation. Titers were determined from A260 with 1.0 absorbance being equivalent to ;1012
particles/ml.
Calcineurin and Acetylcholinesterase mRNA Stability
tional AChE and nAChR indicate that the increased expression of both proteins reflects the availability of their
transcripts (compare Figs. 3 and 5). CsA treatment during
muscle differentiation only augments AChE mRNA levels,
but not mRNA levels of the g-nAChR subunit (Figs. 4 and 5).
The mouse Ache gene has two polyadenylation signals separated by 1.1 kilobase (Li et al., 1993). With a probe that
extends across the first poly A site, we can detect both species. The most 39 signal is in predominant use during differentiation (69–74%) and this percentage is unchanged by CsA
treatment (data not shown). The increased AChE mRNA is
mainly the exon 4 to 6 spliced species because only fully
protected AChE probe (458 bp) was observed after CsA treatment. The inactive analog CsH (Fig. 4) and another immunosuppressive agent tacrolimus (data not shown) were without effect on both AChE and nAChR mRNA levels.
Run-On Transcriptional Rates of Ache Gene Were
Not Altered by CsA. To examine whether the CsA-induced
increases in AChE expression are due to enhanced transcription of the Ache gene, transcriptional rates of Ache and
g-nAchR genes were examined. As indicated in Fig. 6, treatment of 1 mM CsA in differentiating C2–C12 cells did not
affect run-on transcriptional rates of the Ache and g-nAchR
genes.
CsA Increases AChE mRNA Stability in C2–C12
Cells. Because CsA treatment did not enhance run-on transcriptional rates of the Ache gene, the CsA-induced AChE
mRNA expression may be due to increased stability of the
transcripts. To test this hypothesis, rates of AChE mRNA
degradation were examined in cells treated with or without
CsA after blocking transcription with DRB, an adenosine
analog that specifically inhibits RNA polymerase II (Tamm
et al., 1976). As indicated in Fig. 7, the estimated AChE
mRNA half-life in 3-day-differentiated myotubes was ;8 h, a
value similar to that reported previously (Fuentes and Taylor, 1993). CsA treatment increased the estimated half-life of
AChE mRNA to ;16 h.
Precise determinations of mRNA turnover in this system
Fig. 2. Morphology of differentiated
C2–C12 cells treated with CsA or
infected with adenovirus. C2–C12
cells were differentiated for 3 days
in the absence (A) or presence (B) of
1 mM CsA; or infected overnight at
day 1 without (C) or with (D) adenovirus containing the lacZ gene as
described in the text. At day 3 of
differentiation, cells were fixed and
stained for b-gal activity (C and D),
and photos were taken with a 103
phase contrast objective.
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Handschumacher et al., 1984). In contrast, tacrolimus, another immunosuppressive agent active in the subnanomolar
concentration range in other systems (Bierer et al., 1990),
had little influence on AChE expression. Treatment with
rapamycin, a structurally distinct immunosuppressant, in
the subnanomolar concentration range resulted in inhibition
of cell growth and differentiation followed by substantial
reduction of AChE expression (data not shown). Furthermore, treatment with CsH, an inactive analog of CsA, did not
alter AChE expression (Fig. 1).
The effects of CsA are not due to a greater differentiation of
the C2–C12 cells because control and CsA-treated (1 mM, 3
days) cells displayed similar morphology (Fig. 2,A and B). In
addition, muscle creatine kinase activities for differentiating
cells treated with 1 mM CsA for 3 days were similar to control
cells (control: 3.2 6 0.6 A520/ng protein/min; 1CsA: 3.3 6 0.2
A520/ng protein/min, n 5 4). Finally, nAChR expression levels were unchanged (Fig. 3B).
The enhancement of AChE expression by CsA was examined at various stages of muscle differentiation (Fig. 3A).
Fractional increases in AChE expression induced by CsA
were more evident after initial differentiation. However, following wash out of CsA after 1 day of treatment, the increase
in AChE expression was again minimal at day 3. Furthermore, when cells were treated with CsA before terminal
differentiation, no enhancement of AChE expression was observed (data not shown). The increased cellular AChE expression is not due to reduction of AChE secretion because
AChE found in the medium showed a comparable fractional
increase with CsA treatment. The majority of AChE is associated with the cells (data not shown). In contrast to AChE,
cell surface 125I-a-BTX binding sites on the nAChR, whose
increased expression roughly parallels that of AChE upon
differentiation of C2–C12 cells, were not altered by CsA
treatment (Fig. 3B).
As shown in Fig. 4 and summarized in Fig. 5, AChE and
nAChR mRNA increase substantially during muscle terminal differentiation. The parallel sequential increases of func-
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Fig. 3. Kinetics of the influence of CsA on AChE and nAChR expression
during myoblast-to-myotube differentiation in C2–C12 cells. Cells were
differentiated for 3 days in the presence or absence of 1 mM CsA. Cells
from three plates per experimental point were harvested at designated
times and either AChE activity or a-BTX binding sites on the nAChR
were determined. A, AChE activity in C2–C12 cells with or without CsA
treatment. B, a-BTX binding sites in C2–C12 cells treated with or without CsA treatment. AChE activity or a-BTX binding sites on the nAChR
at day 3 of differentiation were chosen as maximum (100%) values.
Values reported are means 6 S.E. from at least six independent determinations.
Influence of Overexpressed Calcineurin Mutants on
AChE Expression. Because cyclophilin is expressed in myoblasts and myotubes at comparable high cellular concentrations, its regulation is less likely to be responsible for the CsA
induction than calcineurin, whose expression associated with
muscle differentiation is enhanced .3-fold. To test this hypothesis, overexpression of constitutively active (HACnADCaM-AI) or dominant negative (HA-BKO) calcineurin
constructs was sought. As indicated in Fig. 2, C and D,
adenovirus infect nearly 100% of the C2–C12 cells and infection does not affect cell differentiation as indicated by the
normal muscle morphology of infected cells. Infected differentiating C2–C12 cells express the calcineurin constructs
well as indicated by expression of a large amount of the HA
peptide (Fig. 9). Muscle morphology also was not altered in
cells overexpressing calcineurin (data not shown). If CsA
enhancement of AChE mRNA level is mediated by calcineurin inhibition, then calcineurin, perhaps through its
phosphatase activity, may play a role in destabilizing AChE
mRNA during myogenesis. If this is the case, overexpression
of HA-CnADCaM-AI should reduce AChE mRNA levels in
differentiating cells, and its effect should be blocked by CsA
treatment. Conversely, overexpression of HA-BKO should
increase AChE mRNA levels in differentiating cells and its
effect should be further enhanced by CsA treatment. These
predictions are generally supported by our findings shown in
Fig. 10. Overexpression of HA-CnADCaM-AI in untreated
differentiating myotubes resulted in a 59% reduction of
AChE mRNA compared with mock-infected, untreated myotubes (P , .001). This calcineurin-induced inhibition was
blocked by CsA treatment (P , .001) at concentrations that
should be expected to inhibit both the endogenous and mutant calcineurins expressed after infection. In contrast, overexpression of HA-BKO in untreated differentiating myotubes
Fig. 4. Representative autoradiogram showing the influence of CsA and
CsH on differentiation-induced expression of AChE transcripts in C2–
C12 cells. Cells were differentiated in the presence or absence of 1 mM
CsA or CsH for 3 days, total RNA was extracted at the indicated times,
and analyzed by RNase protection. The AChE probe extended between bp
1651 and 2107 in the cDNA. Protection by a mRNA species splicing
between exons 4 and 6 should yield a 456-bp band. The receptor probe
extended between bp 1168 and 1716 in the cDNA and the g-nAChR
subunit mRNA should protect a 548-bp species. To standardize for loading differences, a GAPDH probe was included that gave a protected band
of 316 bp. To ensure complete RNA digestion, protection was examined in
the presence of transfer RNA.
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are likely to be confounded by several factors. DRB and
cycloheximide treatments during muscle differentiation promote a superinduction of AChE mRNA suggesting that destabilizing factors with a rapid turnover may influence AChE
mRNA stability in differentiating C2–C12 cells (Fuentes and
Taylor, 1993). The potential for superinduction, along with
the asynchrony inherent in the differentiation process, precludes measurements of mRNA turnover at an earlier stage
when AChE mRNA stabilization may be more evident. Also,
asynchrony of differentiation even at day 4 may result in
multiphasic decay. Finally, the extended half-life of AChE
mRNA in myotubes may be influenced by the generalized
inhibition of transcription. Nevertheless, the enhanced
mRNA levels in the presence of CsA appear to parallel the
diminished initial rate of mRNA degradation and cannot be
accounted for by changes in transcriptional rates.
Intracellular Immunophilin and Calcineurin Levels
in C2–C12 Cells. Certain cell lines are known to be deficient
in tacrolimus binding proteins (Kaye et al., 1992). To ascertain whether the differential effects of CsA and tacrolimus on
AChE expression were due to different levels of intracellular
receptors for these agents in C2–C12 cells, we examined the
concentrations of cyclophilin A, the receptor for CsA, and
FKBP12, the receptor for tacrolimus, in C2–C12 cells. Western blot analyses indicate that myoblasts and myotubes express similar levels of cyclophilin A and FKBP12. However,
calcineurin expression is low in myoblasts, but increases
.3-fold upon muscle differentiation (Fig. 8).
Calcineurin and Acetylcholinesterase mRNA Stability
caused a 48% increase of AChE mRNA compared with mockinfected, untreated myotubes (P , .05), which was further
enhanced by CsA treatment (P , .05) (a total of 140% increase over mock-infected, untreated myotubes), indicative of
CsA possessing the capacity to inhibit the residual calcineurin activity.
Discussion
Fig. 5. Influence of CsA on differentiation-induced expression of AChE
and nAChR mRNAs in C2–C12 cells. Cells were differentiated for 3 days
in the presence or absence of 1 mM CsA, total RNA was extracted, and
mRNA levels for AChE or the g-subunit of nAChR were determined as
described in Fig. 4. The mRNA levels were quantified by taking the ratio
of AChE or nAChR band densities over that of GAPDH to correct for
sample loading errors and then compared with values from 3-day control
samples, which were chosen as 100%. Values reported are means 6 S.E.
from at least nine independent determinations. A, AChE transcript levels
during differentiation. B, g-nAChR subunit transcript levels during differentiation.
Thus, a CsA-sensitive pathway plays a role in the differentiation-induced stabilization of AChE mRNA.
The action of CsA appears to be specific for AChE expression and requires its interaction with intracellular cyclophilins. These conclusions are supported by the following observations. The EC50 of the CsA action (300 nM) correlates well
with the Kd value (200 nM) for its binding to cyclophilins
(Fig. 1; Handschumacher et al., 1984). In addition, CsA treatment selectively enhances expression of AChE, but not
nAChR whose expression is enhanced by transcriptional activation during myogenesis (Evans et al., 1987; Baldwin and
Burden, 1988). Furthermore, the action of CsA is reversible
because treated cells show a normal rate of AChE expression
during myogenesis after CsA was removed (Fig. 3). Finally,
CsH, an inactive analog of CsA, and tacrolimus, another
immunosuppressive agent, neither of which bind to cyclophilins (Handschumacher et al., 1984; Harding et al., 1989;
Siekierka et al., 1989), do not influence AChE expression
(Fig. 1). The inability of tacrolimus to enhance AChE expression is not due to the absence of intracellular receptors as
Fig. 6. Influence of CsA on transcriptional rates of the Ache and nAchR
genes measured by run-on transcription. 32P-a-UTP labeled transcripts
were synthesized in nuclei isolated from C2–C12 cells differentiated for 3
days in the presence or absence of 1 mM CsA and hybridized to slots
containing 5 mg each of the appropriate cDNAs. These cDNAs include
double-stranded g-nAChR and a-tubulin cDNAs in Bluescript II SK (SK)
plasmids and single-stranded AChE antisense cDNA in M13 phage
(M13). Vector DNAs were used as controls. A, representative autoradiogram. B, quantification of the signals by densitometric analysis. Transcriptional rates of the genes in control myotubes were chosen as 100%
values. Densities were normalized with respect to a-tubulin mRNA densities. Values reported are means 6 S.E. from at least three independent
determinations.
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Our findings indicate that treatment of CsA in differentiating C2–C12 muscle cells enhances differentiation-induced
expression of AChE, but not nAChR. The enhancement is
seen at both the level of AChE mRNA and gene product (Figs.
1, 3–5) indicating that CsA primarily influences transcript
production or its stabilization. Previous studies analyzing
run-on transcription rates, transcription rates of Ache-luciferase reporter gene constructs, superinduction of AChE
mRNA by inhibition of protein synthesis, and AChE mRNA
degradation rates all suggest that mRNA stabilization plays
a critical role in enhanced AChE expression associated with
muscle differentiation (Fuentes and Taylor, 1993; Li et al.,
1993; Luo et al., 1994). CsA treatment does not increase the
apparent transcriptional rate of the Ache gene (Fig. 6), suggesting that CsA regulation is due mainly to further enhancement of the stability of AChE mRNA during differentiation. This is confirmed by the finding that CsA treatment
increases the estimated half-life of AChE mRNA (Fig. 7).
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Luo et al.
Fig. 7. Influence of CsA on AChE mRNA stability in C2–C12 cells. AChE
mRNA degradation was measured by RNase protection at different time
points after blocking cellular transcription with 30 mg/ml DRB in differentiating C2–C12 cells treated with or without 1 mM CsA for 3 days. At
this stage, cells have differentiated into myotubes with greatly increased
AChE mRNA. Data are presented as percentage of values from control
myotubes immediately after DRB treatment. The values reported are
means 6 S.E. from four to six independent determinations.
phosphatase activity (Liu et al., 1991). The correlation between differentiation-induced expression of calcineurin (Fig.
8) and AChE (Figs. 3–5) suggests a role of calcineurin in
regulation of AChE mRNA. The down- or up-regulation of
AChE mRNA upon overexpression of the constitutively active or dominant negative calcineurin constructs, respectively, in untreated cells indicates a role for calcineurin in
destabilizing AChE mRNA during myogenesis (Fig. 10). This
regulatory mechanism may serve to control enhanced biosynthesis of AChE during myogenesis, which has a long half-life
of ;50 h (Rotundo and Fambrough, 1980). Because the effects of constitutively active or dominant negative calcineurin on AChE mRNA can be reversed or enhanced, respectively, by CsA treatments (Fig. 10), it is likely that the
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observed for mast cells (Kaye et al., 1992) because FKPB12
can be detected in both myoblasts and myotubes (Fig. 8).
Myoblast differentiation is restricted in G1 phase of the cell
cycle (Clegg et al., 1987). Even though CsA is known to arrest
the cell cycle in G0 phase in cells such as lymphocytes (Schreiber and Crabtree, 1997) and keratinocytes (Karashima et
al., 1996), CsA action on AChE expression is not likely to be
associated with cell cycle arrest because treatment with CsA
before the induction of muscle differentiation does not increase AChE mRNA expression (data not shown). In addition, CsA-treated muscle cells display normal differentiation
as assessed by their muscle fiber morphology, fusion to myotubes (Fig. 2, A and B), creatine kinase activity, and expression of nAChR (Figs. 3–5). Furthermore, when cells were
treated with CsA for 1 day, then allowed to differentiate for
two additional days in CsA-free medium, the AChE expression rate is similar to that of control cells (Fig. 3A), consistent
with CsA action being associated with muscle differentiation,
rather than the immunosuppressant triggering an early
event in differentiation.
It is known that CsA binds to its immediate downstream
target cyclophilin A and inhibits cis-trans proline isomerase
activity of cyclophilin (Harding et al., 1989). However, similar to immunosuppressant activities of this drug (Bierer et
al., 1990), the influence of CsA on AChE mRNA seems independent of the isomerase activity of cyclophilin. This is supported by the findings that expression of cyclophilin A is
independent of muscle differentiation (Fig. 8), whereas the
CsA action appears differentiation-dependent. Furthermore,
overexpression of cyclophilin A does not increase AChE
mRNA levels during myogenesis (Z.D.L., unpublished data).
CsA-cyclophilin and FK506-FKBP complexes are known to
bind to a common target calcineurin and therein inhibit its
Fig. 8. Cellular levels of cyclophilin A, FKBP12, and calcineurin catalytic
subunit in C2–C12 cells at the myoblast and myotube stages. Cell extracts from myoblasts and 3-day differentiated myotubes were subjected
to Western blot analysis with the indicated antibodies as described in
Experimental Procedures. A, representative blots from at least two independent experiments. Positive controls were cyclophilin A and FKBP12
from Jurkat cell extracts (a gift from Dr. Michael Karin, University of
California-San Diego) and purified calcineurin A subunit (Sigma Chemical Co.). B, tabulation of cellular calcineurin A subunit levels. Data
represent means 6 S.E. from three independent determinations.
Calcineurin and Acetylcholinesterase mRNA Stability
Fig. 9. Optimal time for overexpression of calcineurin constructs in
differentiating C2–C12 cells. Cells were differentiated at day 0 and infected overnight with pAdv/HA-CnADCaM-AI as indicated. Cells were
extracted 2 days after infection and subjected to Western blot analysis
with anti-HA antibodies. Uninfected myoblasts (at day 0 of differentiation) and myotubes (differentiated for 3 days) were used as controls.
Bands at 45- and 70-kD positions are backgrounds of the HA antibody
that also are seen in other cells. The HA-CnADCaM-AI protein has a
molecular mass of ;48 kD. Data are representative from two independent infections.
esis. These factors, however, may be influenced by certain
conformations of calcineurin complexes induced by CsA, but
not FK506. In fact, emerging data support an integrated,
calcium-calmodulin-dependent pathway in regulation of
AChE mRNA stability. For example, intracellular immunophilins are found to serve as accessory proteins of ryanodine receptors to modulate intracellular calcium (Timerman
et al., 1993; Brillantes et al., 1994). In addition, calmodulin
regulates intracellular calcium channel activity by interacting with several cytoplasmic domains of the ryanodine receptors (Wagenknecht et al., 1997), which are known to play an
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CsA-induced stabilization of AChE mRNA is mediated
through inhibition of calcineurin. The similar increases of
AChE mRNA in CsA-treated cells with or without overexpression of the calcineurin constructs indicate that CsA at
this concentration (1 mM) may have elicited close-to-maximal
inhibition of endogenous and exogenous calcineurin activities, thus, achieving a near maximal increase in AChE
mRNA (Fig. 10).
The inability of tacrolimus to produce the same response as
CsA (Fig. 1) suggests that the phosphatase activity of calcineurin per se may not be the sole determinant of CsAinduced AChE expression. Specificity for a particular phosphoprotein and/or a downstream association responsive to
the CsA-cyclophilin-calcineurin complex, but not to that of
FK506-FKBP12-calcineurin, may govern the CsA response in
muscle. This hypothesis is supported by the notion that phosphatases exhibit substrate selectivity by recognizing threedimensional patches on phosphoproteins remote from the
dephosphorylation site (Schreiber, 1992). For example, these
drug-immunophilin complexes inhibit phosphatase activity
of calcineurin toward phosphopeptide and phosphoprotein
substrates, yet they cause a 2- to 3-fold activation of phosphatase activity toward a small substrate, p-nitrophenyl
phosphate (Liu et al., 1991). Thus, the catalytic site of the
inhibited calcineurin is still accessible to small substrates,
suggesting that the binding of drug-immunophilin complexes
to an allosteric rather than the active site of calcineurin
modulates substrate selectivity. In addition, CsA-cyclophilin
A and FK506-FKBP12 bind to distinct, highly conserved
regions of calcineurin A (Cardenas et al., 1995), which may
cause distinct conformational changes of the enzyme and
result in different binding affinities for downstream targets.
This behavior was found for phosphatase 2A where replacement of its regulatory subunit by the small T antigen of
simian virus 40 results in alteration of its substrate specificity and cellular localization (Mumby and Walter, 1991).
Collectively, our studies suggest that calcineurin-mediated
dephosphorylation is an important component in the calcium-sensitive regulation of AChE mRNA stability (Luo et al.,
1994, 1996). Dephosphorylation may serve to activate destabilizing factors or inactivate stabilizing factors, which are
involved in governing AChE mRNA lifetimes during myogen-
893
Fig. 10. Influence of overexpression of calcineurin mutants on AChE
mRNA levels. Control and CsA-treated C2–C12 cells were infected with
control adenovirus vectors, or vectors containing a constitutively active
calcineurin construct, pAdv/HA-CnADCaM-AI, or a dominant negative
calcineurin construct, pAdv/HA-BKO, at day 1 of myoblast differentiation
and harvested 2 days later. Total RNA was extracted, treated with
DNase, and subjected to RNase protection. A, representative autoradiogram. B, tabulation of the influence of HA-CnADCaM-AI or HA-BKO
overexpression on AChE mRNA expression. Data represent means 6 S.E.
from 12 independent determinations coming from four separate platings
of C2–C12 cells.
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Luo et al.
important role in the calcium-sensitive regulation of AChE
mRNA stability during myogenesis (Luo et al., 1994). Our
study suggests that calcineurin may be an important mediator of the regulation.
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